Innovare International Journal of Pharmacy and Pharmaceutical Sciences

Academic Sciences ISSN- 0975-1491 Vol 6, Issue 9, 2014

Original Article IN VITRO CULTURE OF BRYUM CORONATUM SCHWAEGR.(BRYACEAE) AND IT’S PHYTOCHEMICAL ANALYSIS

VIJAY KANT PANDEY1, RASHMI MISHRA1, RAMESH CHANDRA1* 1Department of Bio-Engineering, Birla Institute of Technology, Ranchi, India. Email: [email protected] Received: 25 Jul 2014 Revised and Accepted: 26 Aug 2014 ABSTRACT Objective: The purpose of the present investigation was to establish in vitro conditions for moss Bryum coronatum Schwaegr. And to carry out preliminary phytochemical screening of B. coronatum leaf extracts in different solvents. Methods: Fresh unopened, mature capsules were used as explant and surface sterilization of spores bearing capsule with different concentration of sodium hypochlorite (0.5, 1, 2, 4 and 8%) with different time duration. The Murashige and Skoog (MS) medium that contains different concentration(full,1/2, 1/4th, 1/8th strength) with different concentration of sucrose were used for culture this moss. Phytochemical screening were carried out using ethanol, methanol and ethyl acetate leaf extract of B. coronatum to identify various constitutes using the standard procedures. Results: Surface sterilization of spores of this moss was most effective in 4% commercial bleach for 1 min sterilization. The optimum condition for germination of spores and for proper growth of B. coronatum on MS/4 medium strength with sucrose (1.5%), at pH 5.8 and temperature 22±2ºC with 16/8h: light/dark condition. Phytochemical analyses revealed the presence of alkaloids, terpenoids and saponins in all extracts. Conclusion: Four percentage NaOCl aqueous solutions are better for surface sterilization of moss sporophytes. MS/4 medium with 1.5% sucrose found the best medium for spore germination. Solvents extracts showed presence of alkaloids, terpenoids and saponins in all extracts. Keywords: In vitro, Bryum coronatum, Spores, Alkaloids, Terpenoids, Saponins.

INTRODUCTION compound which included chlorophyll, proteins and common sugars and secondary compounds which have terpenoids, alkaloids and Bryophytes (liverworts, and hornworts) are non-vascular phenolic compounds [17]. Terpenoids exhibit various important plants, with nearly 15,000 species worldwide of which pharmacological activities i. e., anti-inflammatory, anti-malarial, approximately 8,000 are mosses, 6,000 are liverworts, and are 200 anticancer, inhibition of cholesterol synthesis, anti-viral and anti- are hornworts [1], was probably reduced early during land plant bacterial activities [18]. Terpenoids are very important in attracting evolution[2,3,4]. Bryophytes possess the following main useful mites and consume the herbivorous insects [19]. characteristics: they have a very simple structure compare to higher plant. Dominant vegetative phase is haploid gametophytes and less Genetic engineering of terpenoid metabolism attracts bodyguards to chromosome number[5]. Ono et al (1988) reported that cell of Arabidopsis. Alkaloids are used as anaesthetic agents and are found bryophytes especially in suspension culture, as ideal materials for in various medicinal plants [20]. morphogenetic, genetic, biochemical, physiological and molecular studies. Bryophytes occur in nearly every ecosystem on earth and B. coronatum Schwaegr. belonging to family Bryaceae (Bryopsida) play a major role in the recycling of carbon and nutrients through which are densely tufted, yellowish-green plant. It stem is short; 5- growth and decomposition[6]. 10 mm long, often branched. Stem leaves imbricate, lanceolate to ovatelanceolate, Setae 1-1.5 cm long, reddish-brown. Its capsules are Nearly 40% of bryophytes are endangered at present and in urgent pendulous, oblong and reddish-brown, the apophysis slightly wider need of active protection and conservation. In nature, bryophytes than the urn and bulging, rounded-obtuse at base, warty when dry. propagated by means of asexual (by multiplication of gametophytes This moss is widespread in tropical to warm-temperate regions Asia, parts) and sexual (through spore) reproduction. Tissue culture Africa, North America, South America, Australia, and Oceanic islands. methods of mosses offer an alternative mean of vegetative It habitat on soil, bricks, cemented bricks and rocks. propagation. The pioneer example for a bryophyte especially moss The aim of this paper is to report establishment of axenic culture of developed into a scientific was moss Physcomitrella moss B. coronatum and to search for the conditions for full plant patens Hedw. [7,8,9,10,11]. The main advantage of P. patens is the development. Such knowledge is essential for designing ex situ easy way of its axenic in vitro cultivation on solid mineral conservation schedules, for future introduction and reintroduction medium[12], in liquid culture in flasks and in [13-14]. Unlike and for evaluationof the biotechnological potential. For development other higher plant, in vitro culture of mosses cannot be initiated by using of protocol for the full of B. coronatum. Our data will leaf explants since surface sterilization causes cell death. contribute future conservation biology of this moss as well as other Phytochemicals are naturally occurring in plants, leaves and other related mosses. Preliminary phytochemical screening of B. vegetative parts and roots. These phytochemicals have a role in coronatum leaf extracts in different solvents like ethanol, methanol defense mechanism of plants and in protection of plants from and ethyl acetate is being reported. various diseases. Chemical constituents and ethno-bryology of MATERIALS AND METHODS bryophytes are not elaborated very well [15-16]. Bryophytes are extremely rich in phenols (flavonoids and bibenzyl derivatives), Fully developed plant of B. coronatum with sporophytes and terpenoids, glycosides, fatty acids and also some aromatic gametophyte were collected from BIT, Mesra campus, Ranchi, India compounds. It also considered as a remarkable reservoir of new, in spring of 2013. Fresh unopened, mature capsules were used as natural products or secondary compounds, many of which have the starting plant material. Collected plant materials were identified shown interesting biological activities. Phytochemicals are primary by Botanical survey of India (BSI), Kolkata, India. For this voucher

Pandey et al. Int J Pharm Pharm Sci, Vol 6, Issue 9, 307-311 specimen were deposited as plant herbaria in BSI, Kolkata and also well in ice. Sulphuric acid was then added carefully. A colour change in department of Bioengineering, BIT Mesra. Collected plants with from violet to blue to green indicates the presence of a steroidal healthy, immature sporophytes that contain spores wash them nucleus (that is, a glycone portion of glycoside). thoroughly in running tap water for 5 min. Immature capsules of B. coronatum with operculum or both with operculum and calyptra, Tests for steroids and undamaged sporophytes were separated from the i. A red colour produced in the lower chloroform layer when 2 ml of gametophytes and washed in distilled water. In vitro cultures of B. or ethanol, methanol and ethyl acetate leaf extracts of moss was coronatum were initiated by surface sterilization of spores bearing dissolved in 2 ml of chloroform and 2 ml concentrated sulphuric acid capsule with different concentration of sodium hypochlorite (0.5, 1, was added in it, indicates the presence of steroids. 2, 4 and 8%) with different time duration. Then, rinsed them 3 times in cold, sterile distilled water for 5 min. Capsules were opened with ii. Development of a greenish colour when 2 ml of the organic extract a sterile needle and transferred onto solid medium. Capsules were was dissolved in 2 ml of chloroform and treated with sulphuric and opened aseptically under the laminar flow cabinet and spores were acetic acid indicates the presence of steroids. transferred with a sterile needle to petri dishes containing 20 ml solid medium (MS medium with different concentrations such as RESULTS AND DISCUSSIONS 1/2, 1/4th, 1/8th and its normal strength with and without sucrose. In vitro culture of profits usually started from sporophytes via spore The basal medium (BM) contained MS(Murashige and Skoog)[21] germination [22]. However in some species like Rhodobryum,spores mineral salts and vitamins, 100 mg/L myo-inositol, 0.70% (w/v) are not regularly available and also some species of bryophytes do agar (Bacteriological) without any supplements of growth not produce sporophytes regularly. For the establishment of axenic regulators. The medium pH was adjusted to 5.8 prior to autoclaving culture from gametophytes and sporophytes of bryophytes there is at 121ºC for 20 min. Cultures were grown at 22±2ºC under white effective concentration of commercial bleach for surface sterilization fluorescent tubes at photon fluorescent rate of 2s and a which to kill the xenic organisms on the plants and not to harm the day/night regime of 16/8h. Moss plants were sub-cultured after 1 plants at the same time[22,23,24,25]. Surface sterilization of the month interval. 47 μmol/m sporophytes was more successful since we choose the almost mature but unopened capsules. The higher concentration of Preparation of plant extract commercial bleach harms the spores which cause decrease in spores The leaves of B. coronatum was removed from the plants and then germination. Gametophytes of this moss are very delicate because of washed with distilled water to remove any adhering soil or a very thin cuticle so it does not survive even in short time surface extraneous material and were air dried inside the room at room sterilization with commercial bleach at various concentrations. High temperature (28-32 for 7day until water content of the sample concentration of commercial bleach is lethal for gametophytes of become negligible and then grinded to coarse power by mechanical this moss. grinder. Methanol, ethanol˚C) and ethyl acetate extracts of this sample was prepared by soaking 2 g dried power sample in 20 ml of For in vitro establishment of mosses, the first phase was respective solvents for 24 hrs. Extracts were filtered and evaporated multiplication and propagation, followed by reintroduction of the under reduced pressure. cultured specimens to native and potentially suitable habitats. It was found that surface sterilization of sporophytes by various Phytochemical screening concentration of commercial bleach was effective but comparatively less effective for the gametophytes. It was found that survival Chemical tests were carried out using ethanol, methanol and ethyl percentage was increasing with increase in commercial bleach acetate leaf extracts of B. coronatum to identify various constituents concentration (0.5%-4%), then it deceased with further increase in using the standard procedures as described by Sofowara (1993), commercial bleach concentration. Sporophytes of B. coronatum were Trease and Evans (1989) and Harborne (1973). treated for 1 min in all cases. However contamination percentage Test for Alkaloid decreased with increase in commercial bleach. Maximum survival rate of sporophytes was found with 1 min treatment of 4% Three ml leaf extractof moss was stirred with 3 ml of 1% HCl on commercial bleach(Fig 1). steam bath. Mayer and Wagner’s reagent was then added to mixture. Turbidity of the resulting precipitate was taken as an evidence for Asexual gametophytes were used as explant, although they were the presence of alkaloid. mostly vulnerable to disinfectants. The cutting treatment of protonema nearly doubles the biomass in short time [26], but sub- Test for Tannins culture is necessary. The differentiation into gametophores and protonemal growth depend not only on internal factor transported About 2 ml leaf extract of moss was stirred with 2 ml of distilled from cell to cell, but also on interactions with substrate [27]. Spore water and few drops of FeCl3 Solution were added. Formation of germination in B. coronatum in in vitro cultures occurred after 5-6 green precipitate was indication of presence of tannins. days of establishment of axenic cultures of this moss species. The Test for Saponins culture media for the germination of this moss was MS media that was solidified by 0.7% agar. The germination of moss was done in 5 ml of leaf extract of moss was shaken vigorously with 5 ml of different concentration of MS media (Full, 1/2, 1/4 & 1/8) and also distilled water in a test tube and warmed. The formation of stable with different concentration of sucrose (0%, 1%, 1.5% & 3%). It was foam was taken as an indication of the presence of saponins. found that germination of spores of B. coronatum was good in 1/4 strength of MS medium with different concentration of sucrose. First Test for Flavonoids the percent of germination increased from full strength to 1/4 To 1 ml of leaf extract of moss, 1 ml of 10% lead acetate solution was strength then decreased in 1/8MS medium with different sucrose added. The formation of a yellow precipitate was taken as a positive concentration. Maximum germination rate was found in 1/4MS with test for flavonoids. 1.5 % sucrose concentrations in continuous light. No spore germination was occurred in absolute dark. Test for Terpenoids The vegetative development of the gametophyte in mosses involved 2 ml of leaf extract of moss was dissolved in 2 ml of chloroform and two stages: the filamentous protonema stage, and the budding evaporated to dryness. 2 ml of concentrated sulphuric acid was then gametophore stage that involves more complex cell differentiation. added and heated for about 2 min. Development of a greyish colour Spores of B. coronatum shown in figure 3. These spores turned indicates the presence of terpenoids. bright green after 3–4 days of inoculation of spore on 1/4MS Tests for glycosides: Liebermann’s test medium in continuous illumination as well as in alternate light and dark conditions and develop early filamentous protonema having 2 ml of the leaf extract of moss was dissolved in 2 ml of chloroform chloronema (Fig. 3B) and after 7-8 days they germinated to produce and then 2 ml of acetic acid was added in it. The solution was cooled branched chloronema with abundant rounded chloroplasts. After 10

308 Pandey et al. Int J Pharm Pharm Sci, Vol 6, Issue 9, 307-311 days of spore germination, the distal end of chloronema started to is complex. It is expected that sucrose have positive effects on become hyaline on account of disappearance of chloroplasts but only biomass increase. As it does in bryophytes species and higher plant a few chloronemal filaments were differentiated into conspicuous species[31-32] but in some species sucrose had very little and no caulonema (brown coloured filaments having a few spindle shaped effect on bud germination. chloroplasts and oblique septa).

Fig. 1: Survival and contamination percentage of B. coronatum spores in culture. Note: Error bar shows standard deviation.

Fig. 3: In-vitro culture of Bryum coronatum Schwaegr. (Note: Scale bar equals 100 µm in Fig A, B & C and 1 cm in Fig D, E & F)

Fig. 2: Spore germination rate of B. coronatumin different MS Bryum species do not require any critical photoperiod for the onset media with different Sucrose concentrations after 5-6 day of of the reproductive phase[33] but while working on this B. inoculation. coronatum it was found 16/8: day/dark periods. Kumar and Chopra (1983) reported that the response of Bryum species increase with Note: Error bar shows standard deviation increasing temperature upto 24

optimum temperature for growth of B. coronatum was 22±2 Chopra and Kumra (1988) reported˚C, although that some it mosswas foundspecies that could The culture contained mainly green chloronema and a few not germinate or even grow without a symbiosis with fungi and˚C. caulonema or hyaline chloronema are used for sub-culture on same bacteria. In this experiment a very easy, effective and convenient media. Protonemal colonies were develop after subculture of method for surface sterilization and in vitro culturing of moss B. protonema on 1/4MS medium after 30 day of inoculation (Fig. 3D). coronatum is shown. Further testing is needed, with more After 45 – 50 days of inoculation, protonemal buds were repetitions, with different range of growth regulator concentrations differentiated from the main chloronemal filaments or basal cells of along with observation times, to determine what are the effect of the the chloronemal branches and distributed throughout the growth regulators for germination and differentiation of protonemal patch in a scattered manner (Fig. 3E). Protonemal buds gametophytes. subsequently developed into young gametophytes (Fig. 3F) in form of erect main axis with numerous, more crowded erects leaves and The phytochemical characteristics of the leaf extract of B. coronatum brown colour rhizoids. 1/4MS medium with 1.5% sucrose gave investigated are summarized in table-1. Phytochemical analyses abundant growth of protonema and rhizoids. In 65-70 day-old revealed the presence of alkaloids, terpenoids and saponins in all culture, well developed population of erect gametophytes developed samples. Methanol and ethyl acetate extract of this moss plant also (Fig. 3F). Rhizoids were morphologically similar to caulonema being have steroid while in ethanol extracts doesn’t have steroids. Ethanol, brown in colour and with oblique septa but devoid of plastids. It has methanol extracts also have glucosides while this compound was been reported previously for other bryophytes that the pattern of absent in ethyl acetate extract. The alkaloids contained in plants are protonema development in in vitro culture varies depending on the used in medicine as anaesthetic agents [34]. The presence of mineral media[28]. In vitro culture of the moss Bryum argenteum saponins in plants have been reported to be responsible for the tonic was established from sterilized spores and its gametophytes grown and stimulating activities observed in Chinese and Japanese medical in in vitro were used to investigate the influence of different herbs[35]. Different phytochemicals have been found to possess a substances on secondary protonema and on the growth and wide range of activities, which may help in protection against multiplication of the gametophytes [25]. Despite the importance of chronic diseases. For example,alkaloids protect against chronic the mineral medium, interactions between its components and diseases. Saponins protect against hypercholesterolemia and organic compounds, such as sugar, may affect culture development antibiotic properties. Steroids and triterpenoids show the analgesic and growth [28-29]. Vitamins also appear to be important properties. The steroids and saponins were responsible for central determinants of various physiological processes in higher plants nervous system activities. Steroidal compounds are of importance [30]. A number of significant effects of sucrose on culture and interest in pharmacy and pharmaceutical science because of development were observed. Though, interpretation of these effects their relationship with compounds such as sex hormones [36].

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Methanol and ethyl acetate extract of this plant also have steroid 3. Graham LE, Cook ME, Busse JS. The origin of plants: body plan while in ethanol extracts doesn’t have steroids. Ethanol, methanol changes contributing to a major evolutionary radiation. Proc extracts also have glucosides while this compound was absent in Natl Acad Sci USA 2000;97:4535‐40. ethyl acetate extract. Previous reports convey that br yophytes 4. Taylor TN, Kerp H, Hass H. Life history biology of early land possess medicinal value [37] and are good sources of antibiotics plants: deciphering the gametophyte phase. Proc Natl Acad Sci [38]. USA 2005;102:5892‐97. 5. Glime JM. Methods in Bryology. Mainz: Proc Bryol Meth. The preliminary result on moss plant under study (B. coronatum) Workshop 1988;1-16:99-105. has also shown antimicrobial activity and its detailed investigation is 6. Vanderpoorten A, Goffinet B. Introduction to bryophytes. going on our lab. Cambridge: University Press; 2009. p. 26-42, 185-213. 7. Reski R. Development, genetics and molecular biology of mosses. Bot Acta 1998;111:1‐15. Table 1: Phytochemical screening of B. coronatum Schwaegr. in 8. Reski R. as a novel tool for plant

different solvents extracts functional genomics. In: Vasil IK. (ed) Plant biotechnology 2002 Chemical Constitutent Ethanol Methanol Ethyl acetate and beyond. Kluwer Acad Publ; 2003. p. 205‐9. Extract extract Extract 9. Cove DJ, Bezanilla M, Harries P, Quatrano RS. Mosses as model Alkaloid + + + systems for the study of metabolism and development. Annu Tannins - - - Rev of Plant Biol 2006;57:497‐520. Saponins + + + 10. Beike AK, Decker EL, Frank W, Lang D, Vervliet-Scheebaum M, Flavanoid - - - Zimmer AD, Reski R. Applied Bryology Botechnology. Trop Glycosides + + - Bryol 2010;31:22‐32. Steroid - + + 11. Prigge MJ, Bezanilla M. Evolutionary crossroads in Terpenoids + + + developmental biology: physcomitrella patens. Development 2010;137:3535‐43. Note: +: Present and -: Absent 12. Reski R, Abel WO. Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using iso‐pentenyladenine. Planta 1985;165:354‐58. 13. Decker EL, Reski R. The moss bioreactor. Curr Opin Plant Biol CONCLUSION 2004;7:166‐70. Surface sterilization through commercial bleach (4% NaOCl) is 14. Hohe A, Reski R. From axenic spore germination to molecular easier to achieve on sporophytes than gametophytes. Maximum farming: one century of bryophyte in vitro culture. Plant Cell survival rate of sporophytes was found with 1 min treatment of 4% Rep 2005;23:513‐21. commercial bleach. The best condition for germination of spores and 15. Sabovljevic A, Sabovljevic M. Bryophytes, a source of bioactive in vitro propagation of plant in B. coronatum on MS/4 medium and new compounds, in Recent Progress in Medicinal Plants strength with sucrose (1.5%), at pH 5.8 and temperature 22 Vol. 22:Phytopharmacology and Therapeutic values IV, 16/8: light/dark condition. Higher sucrose concentrations (>3%) in Studium Press LLC, TX; 2008. p. 9-25. MS medium tended to have a negative effect on germination˚C with of 16. Sabovljevic A, Sokovic M, Sabovljevic M, Grubisic D. spores. 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Genetic engineering of terpenoid metabolism attracts methanol and ethyl acetate leaf extracts of B. coronatum were bodyguards to Arabidopsis. Sci 2005;309:2070-72. investigated for phytochemical screening. It analyses revealed the 20. Herouart D, Sangwan RS, Fliniaux MA, Sangwan-Norreel BS. presence of alkaloids, terpenoids and saponins in all samples. Variations in the Leaf Alkaloid Content of Androgenic Diploid Methanol and ethyl acetate extract of this plant also have steroid Plants of Datura innoxia. Planta Med 1988;54:14-7. while in ethanol extracts doesn’t have steroids. Ethanol and 21. Murashige T, Skoog F. A revised medium for rapid growth and methanol extracts also have glucosides while this compound was bioassays with tobacco tissue culture. Physiol Plant absent in ethyl acetate extract. 1962;15:473. 22. Sabovljevic M, Bijelovic A, Dragicevic I. In vitro culture of CONFLICT OF INTERESTS mosses: Aloina aloides (K. F. Schultz) Kindb, Brachythecium velutinum (Hedw. ) B. S. &G. , Ceratodon purpureus (Hedw. ) Declared None Brid. , Eurhynchium praelongum (Hedw. ) B. S. &G. and Grimmia ACKNOWLEDGEMENT pulvinata (Hedw. ) Sm. Turk J Bot 2003;27:441-46. 23. Vujicic M, Sabovljevic A, Sabovljevic M. Axenically culturing the Authors acknowledge the support got from UGC MRP Project F No. bryophytes: a case study of the moss Dicranum scoparium Hedw. 37-116/2009 (SR), Birla Institute of Technology, Department of Bio- (Dicranaceae, Bryophyta), Botanica Serbica 2009;33(2):137-40. Engineering and Centre of Excellence (COE) TEQUIP -II- for 24. Vujicic M, Cvetic T, Sabovljevic, Sabovljevic M. Axenically providing R&D facilities and Director, National Botanical Research culturing the bryophytes: a case study of the liverwort Institute, Lucknow (CSIR)and Botanical survey of India (BSI), Marchantia polymorpha L. ssp. Ruderalis Bischl. & Boisselier Kolkata, India for providing information related to identification (Marchantiophyta, Marchantiaceae). 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